Mineralogía, Petrografía y Análisis Microstratigráfico

Described above, the post column connection to the “pigtail” was originally performed by a piece of fluoropolymer tubing manually drilled to 360 μm I.D. During the gradient storage

loop analysis, the PicoClearTM _{union from New Objective was introduced to the lab. Used}

previously for UV detection studies, this union was compared to the prior fluoropolymer tube union for mass spectral analysis. Using Setup 2 (Figure 3.13), the same standard digest of BSA (bovine serum albumin) was processed using the fluoropolymer tubing connection and by the

PicoClearTM _{union. Both unions were placed at the end of the column heater, which was at 65°C.}

To accommodate the instantaneous release of pressure from the system shown in Setup 2 (Figure 3.13), a bleed valve was employed (Figure 3.15). The bleed valve consisted of a 40 kpsi mechanical pin valve connected to a low volume tee. The valve was operated via a solenoid valve controlled by the nanoAcquity software. After the chromatographic run was complete, this valve was opened and the pressure was released slowly. The outlet of the pin valve was connected to 1 m of 20 μm I.D. capillary.

3.3 Results and Discussion

3.3.1 Freeze/Thaw Valve

After the freeze thaw valve was manufactured, it was tested (Figure 3.11) to determine

the pressure limitations. Originally the temperature controller was set to -50°C with a ± 20°C

dead band. A mixture of 50:50 acetonitrile:water was tested and a 50 μm I.D. capillary (360 μm O.D., 1 m in length) was placed in the FT valve. Preliminary studies indicated that as the

diameter of the capillary increases beyond 50 μm, the frozen plug cannot seal against high

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Once frozen, the pressure on the pneumatic amplifier was slowly ramped to 30 kpsi. However, on the UHPLC system the pressure does not slowly ramp, it is instantly initiated. Therefore, this was repeated except the pressure was instantly applied at 30 kpsi to the FT valve. The valve successfully held for 30 min at 30 kpsi in 50:50 water:acetonitrile, both with ramped and instant pressure application. Both of these conditions (ramped and instant pressure) were also tested at 60 kpsi and held successfully for 30 min each.

Next, 99.5:0.5 (water:acetonitrile) was tested and the capillary was flushed with the new mobile phase. Although this mobile phase composition held for 30 min at 30 kpsi (both with ramped and instant pressure application), it did not at 60 kpsi. The dead band to the

temperature controller was therefore lowered to ± 10°C. This initiated cryogen flow when the

temperature rose above -40°C instead of the previous dead band setting of -30°C. With this

mobile composition consisting mostly of water it was surprising that -30°C did not hold at 60

kpsi. Once the dead band was lowered, 99.5:0.5 held consistently at 60 kpsi when ramped and when instant pressure was applied. The final mobile phase composition tested was 15:85

(water:acetonitrile). Keeping the same temperature control settings (-50 ± 10°C) this mobile

phase combination held at 30 kpsi and 60 kpsi for both ramped and instantaneously applied pressure. Since 60 kpsi was the highest pressure available for testing within the lab, and it is ~30 kpsi greater than the UHPLC’s routine operating pressure, these tests validated

implementation of the FT valve into the UHPLC system.

The time required to operate the FT valve was monitored through this process. Initially,

taking the FT valve from room temperature to -50°C required the cryogenic valve to remain

open for ~ 5 sec. After this “initial freeze” was performed, intermittent exposures to liquid CO2

required 1-2 sec. The time lasting between freezes was approximately 2.5-3 min when the

temperature controller set point was -50°C. The 5 seconds required to initially freeze/close the

valve greatly reduced the time from the previous Peltier system (2.5 min),17 _{and previous}

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Up to this point, thawing of the valve consisted of turning off the temperature controller and allowing room temperature to thaw the system. This typically required between 5-7 min. However, as shown in Figure 3.9, a power supply and thermoelectric heater were installed to accelerate the thawing process. The output temperature of the copper cup when placed on the thermoelectric heater was recorded with applied power (Figure 3.16). When 0.4 W of power was applied for 10 sec, the valve completely thawed in 1 min. Although the application of the

thermoelectric heater did greatly decrease the time required to thaw the system, it was rarely utilized. Most of the run times for meter long columns on the UHPLC system are 120-140 min. Thus, waiting 5-7 min for room temperature to thaw the FT valve is more than reasonable. However, if shorter run times are sought, the thermoelectric heater can be easily activated to reduce thawing time.

With the FT valve successfully tested, it was initially placed behind the high pressure isolation mechanical pin valve (Figure 3.6). The FT valve was to serve as a support valve to the high pressure isolation valve, since it was this mechanical pin valve that leaked and was

damaged most often. Also, the high pressure isolation valve could immediately stop flow so the FT valve could freeze the immobile liquid (Figure 3.17a). It was soon discovered that if the flow rate from the nanoAcquity was stopped, the FT valve did not require the presence of the high pressure isolation valve to halt flow and could efficiently freeze the liquid inside the capillary itself. Thus, although originally intended to support the high pressure isolation valve, the FT valve eventually replaced it altogether, and later replaced the high pressure vent valve as well (Figure 3.17, b and c). These configurations were compared to the original mechanical pin valve system (Figure 3.1) and the resulting chromatograms are shown in Figure 3.18. A sample of digested bovine serum albumin (BSA) with a gradient of 4-40% in 100 min on a 109 cm 1.7 μm BEH column (~4% change per column volume) was processed on all valve configurations. The flow rate in all configurations was ~300 nL/min and no leaks were suspected for the mechanical pin valve system.

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As shown in Figure 3.18, the region of the gradient wash (~110-150 min) contains large broad peaks when the mechanical pin valve system was used. These peaks were no longer present when the FT valve was installed (Figure 3.18, a-c). When the FT valve was the only valve source (Figure 3.18a) peaks were clearly detectable in this section of the chromatogram. More peaks were also present in the beginning of the chromatograms when applying the FT valve (18- 30 min). The peak capacity of the BSA sample for the mechanical pin valve system was 201 and for all cases where the FT valve was used the peak capacity increased > 240. Therefore,

preliminary studies indicated that the FT valve could hold pressures up to 60 kpsi and provide improved chromatographic results compared to the prior mechanical pin valve configuration.